1. Field of the Invention
The present invention concerns the field of genetic engineering and more particularly the discovery of a unique light-regulated transcription factor that can be used to control the flowering time of plants.
2. Background of the Invention
The Sun is the primary source of energy on the Earth. It is obvious that impinging solar energy warms our atmosphere and drives the Earth""s climate. Perhaps less obvious is that virtually all biological energy including the xe2x80x9cfossilxe2x80x9d fuels that power our civilization are solar in origin. Solar energy is captured for biological use by photosynthesis, a metabolic process that occurs in green plants. During photosynthesis light energy is captured in various chemical compounds that provide food for all nonphotosynthetic organisms.
Since green plants essentially xe2x80x9cfeedxe2x80x9d on light, it comes as no surprise that these organisms are exquisitely sensitive to light. Many people are aware that plants grow towards a light source in an effort to receive sustaining illumination. However, a green plant""s responsiveness to light is much more complex than merely growing towards a light source. Plants contain complex systems for actually measuring the duration of day and night lengths so as to synchronize their growth and lifecycles with the seasons. It is these timing processes that cause chrysanthemums to flower in the autumn and other ornamental and crop plants to flower and fruit at characteristic times. Clearly, the ability to accurately control flowering to promote it or delay it as necessary would be of great economic value. In the middle decades of this century a tremendous amount of biological research was carried out in search of the ever elusive xe2x80x9cflowering hormonexe2x80x9d orfiorigen which, for a time, was the holy grail of plant physiology. Although the quest for florigen ended in failure, much was learned about how plants perceive and respond to environmental factors such as the seasonal changes in day length.
Although green plants have multiple light receptors, the protein-pigment phytochrome has been shown to be the primary receptor by which plants track day length and orchestrate a number of light-regulated responses. Phytochrome is a chromoprotein formed by combining a linear tetrapyrolle pigment with an apoprotein. As such it shows some similarities to phycobiliproteins which are accessory pigments of certain algae and photosynthetic bacteria. Phytoclirome has the somewhat unusual property of existing in two different photochemically interconvertible forms know as Pr (phytochrome-red) and Pfr (Phytochrome-far red). Phytochrome is synthesized in the Pr form which has an absorption maximum in the red region of the optical spectrum. Numerous experiments have shown that the Pr form of phytochrome is essentially inactive in terms of eliciting changes in plant metabolism. However, when Pr absorbs red light (R), it is rapidly converted into the active Pfr form. Pfr has an absorption maximum in the far red (near infrared) portion of the optical spectrum. Absorption of far red light (FR) induces a back conversion of Pfr to inactive Pr. This red-far/red interaction provides a powerful test of whether a given plant response is phytochrome mediated. For example, if dark-grown (etiolated) seedlings are briefly exposed to red light, Pfr will be formed and there will be a concomitant response. However, if the red light exposure is quickly followed by a far-red light exposure (which converts Pfr to inactive Pr) the response will be prevented. The reversibility of a red light response by a far-red light exposure is a hallmark of a phytochrome response.
Although much is known about the phytochrome proteins and their encoding genes, relatively little is known about how the Pfr effects a response in the plant. Many plant genes are light-regulated and that at least some of this regulation is controlled or influenced by phytochrome. Among the genes whose expression is either negatively or positively influenced by phytochrome are several that have been shown to be transcriptionally regulated. These genes include those encoding the small subunit of ribulose bisphosphate carboxylase/oxygenase, the major light-harvesting chlorophyll a/b binding-proteins (Lhcb) of Photosystem II, NADPH: protochlorophyllide oxioreductase, ferredoxin and phosphoenolpyruvate carboxylase, all components of photosynthesis. While the promoter regions are known for many of these genes, the transcriptional factors that bind to these nucleic acid regions are generally unknown. Furthermore, the signal transduction pathways connecting Pfr to these transcriptional factors are largely unknown. These matters have been recently reviewed in Tobin, E. M. and Kehoe D. M. xe2x80x9cPhytochrome regulated gene expression,xe2x80x9d Seminars in Cell Biology 5: 335-46 (1994) to which the reader is directed for more detailed information.
The present invention involves the isolation and characterization of the first discovered phytochrome-regulated transcriptional factor, a protein designated CCA1 which binds to the promoter region of a chlorophyll binding protein gene (Lhcb1*3) of Arabidopsis. The Lhcb1*3 gene of Arabidopsis is known to be regulated by phytochrome in etiolated seedlings where a brief illumination by red light results in a large increase in the level of mRNA from this gene. Karlin-Neumann, G. A., Sun, L., and Tobin, E. M. Plant Physiol. 88:1323-31 (1988). A DNA binding activity, designated CA-1, that interacts with the promoter region of Lhcb1*3 was discovered in cellular extracts. Sun, L., Doxsee, R. A., Harel, E., and Tobin, E. M., Plant Cell 5: 109-21 (1993) (Sun et al., 1993). The promoter region to which CA-1 binds has been shown to be necessary for normal phytochrome regulation of the Lhcb1*3 gene. Kenigsbuch, D. and Tobin, E. M. Plant Physiol. 108:1023-27 (1995). Modification of the expression of CCA1 using techniques of genetic engineering results in unexpected changes in the timing of plant flowering.